Process for the preparation of D-pantothenic acid and/or salts thereof

ABSTRACT

The invention provides a process for the preparation of D-pantothenic acid and/or salts thereof or feedstuffs additives comprising these by fermentation of microorganisms of the  Bacillus  group, in particular those which already produce D-pantothenic acid, which comprises enhancing, in particular over-expressing, in the microorganisms one or more of the nucleotide sequence(s) which code(s) for the gene or ORF ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqjK and yqhI.

The instant application is a 371 of PCT/EP02/05962 filed on May 31, 2002 and claims the benefit of U.S. provisional application 60/305,143 filed on Jul. 16, 2001 and German application 10133161.4 filed on Jul. 07, 2001.

FIELD OF THE INVENTION

This invention relates to a process for the preparation of D-pantothenic acid and/or salts thereof or mixtures comprising these using microorganisms of the Bacillus group in which at least one or more of the genes or open reading frames (ORF) chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK And yqhI is or are enhanced.

PRIOR ART

Pantothenic acid is produced worldwide in an order of magnitude of several thousand ton a year. It is used inter alia in human medicine, in the pharmaceuticals industry and in the foodstuffs industry. A large portion of the pantothenic acid produced is used for nutrition of stock animals such as poultry and pigs.

Pantothenic acid can be prepared by chemical synthesis, or biotechnologically by fermentation of suitable microorganisms in suitable nutrient solutions. In the chemical synthesis, DL-pantolactone is an important precursor. It is prepared in a multi-stage process from formaldehyde, isobutylaldehyde and cyanide, and in further process steps, the racemic mixture is separated, D-pantolactone is subjected to a condensation reaction with β-alanine, and D-pantothenic acid is obtained in this way.

A typical commercial form is the calcium salt of D-pantothenic acid. The calcium salt of the racemic mixture of D,L-pantothenic acid is also customary.

The advantage of the fermentative preparation by microorganisms lies in the direct formation of the desired stereoisomeric form, that is to say the D-form, which is free from L-pantothenic acid.

Various species of bacteria, such as e.g. Escherichia coli (E. coli), Arthrobacter ureafaciens, Corynebacterium erythrogenes, Brevibacterium ammoniagenes, Corynebacterium glutamicum, Bacillus subtilis and also yeasts, such as e.g. Debaromyces castellii can produce D-pantothenic acid.

Instructions for improving the fermentative production processes are, for example, EP-A 0 493 060, EP-A-0590857, U.S. Pat. No. 5,518,906, WO97/10340, WO01/21772 or U.S. Pat. No. 6,184,007.

After fermentation, the D-pantothenic acid or the corresponding salt is isolated from the fermentation broth and purified (EP-A-0590857 and WO96/33283). The fermentation broth-containing D-pantothenic acid can also be dried with the biomass produced during the fermentation (U.S. Pat. No. 6,238,714) and then used in particular as a feedstuffs additive.

OBJECT OF THE INVENTION

The inventors had the object of providing new measures for improved fermentative preparation of D-pantothenic acid and/or salts thereof, and animal feedstuffs additives comprising these.

SUMMARY OF THE INVENTION

When D-pantothenic acid or pantothenic acid or pantothenate are mentioned in the following text, this means not only the free acids but also the salts of D-pantothenic acid, such as e.g. the calcium, sodium, ammonium or potassium salt.

The invention provides a process for the preparation of D-pantothenic acid and/or salts thereof using microorganisms of the Bacillus group which in particular already produce D-pantothenic acid and in which at least one or more of the nucleotide sequence(s) which code(s) for the ybbT-ORF, ywkA-ORF, yjmC-ORF, ytsJ-ORF, mdh gene, cysK gene, iolJ gene, pdhD gene, yuiE-ORF, dhaS gene, adk gene, yusH-ORF, yqhJ-ORF, yqhK-ORF and yqhI-ORF is or are enhanced, in particular over-expressed.

In particular, the process is a process which comprises carrying out the following steps:

-   -   a) fermentation of microorganisms of the Bacillus group in which         at least one or more of the genes or open reading frames chosen         from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK,         iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK: and yqhI is or         are enhanced, in particular over-expressed, optionally in         combination with the attenuation or enhancement of further genes         or open reading frames,     -   b) optionally in the presence of alkaline earth metal compounds,         these being added to the fermentation broth continuously or         discontinuously in preferably stoichiometric amounts     -   c) concentration of the D-pantothenic acid or the corresponding         salts in the medium or the fermentation broth or optionally in         the cells of the microorganisms of the Enterobacteriaceae family         and     -   d) after conclusion of the fermentation, isolation of the         D-pantothenic acid, and/or of the corresponding salt(s).

The invention also provides a process in which, after conclusion of the fermentation, all or some (0 to 100%) of the biomass remains in the fermentation broth, and the broth obtained in this way is processed, optionally after concentration, to a solid mixture which comprises D-pantothenic acid and/or salts thereof and optionally comprises further constituents of the fermentation broth.

DETAILED DESCRIPTION OF THE INVENTION

The term “enhancement” in this connection describes the increase in the intracellular activity of one or more enzymes or proteins in a microorganism which are coded by the corresponding DNA, for example by increasing the number of copies of the gene or genes, of the open reading frame (ORF) or ORFs, using a potent promoter or a gene or allele or ORF which codes for a corresponding enzyme or protein with a high activity, and optionally combining these measures.

Open reading frame (ORF) describes a section of a nucleotide sequence which codes or can code for a protein or polypeptide or ribonucleic acid to which no function can be assigned according to the prior art. After assignment of a function to the nucleotide sequence section in question, it is in general referred to as a gene.

By enhancement measures, in particular over-expression, the activity or concentration of the corresponding protein is in general increased by at least 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400% or 500%, up to a maximum of 1000% or 2000%, based on that of the wild-type protein or the activity or concentration of the protein in the starting microorganism.

The microorganisms which the present invention provides can produce D-pantothenic acid from glucose, sucrose, lactose, fructose, maltose, molasses, starch, cellulose or from glycerol and ethanol. They are representatives of the Bacillus group, in particular of the genus Bacillus, preferably the species Bacillus subtilis.

The Bacillus group includes, inter alia, Bacillus subtilis, Bacillus lentimorbus, Bacillus lentus, Bacillus firmus, Bacillus pantothenticus, Bacillus amyloliquefaciens, Bacillus cereus, Bacillus circulans, Bacillus coagulans, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, Bacillus thuringiensis, Bacillus halodurans, Bacillus brevis, Bacillus stearothermophilus Bacillus, and other so-called group 1 Bacillus species which are characterized by the corresponding 16S rRNA type (Priest (1993), In: Bacillus subtilis and Other Gram-Positive Bacteria, eds. Sonenshein et al., ASM, Washington, D.C., USA).

Suitable D-pantothenic acid-producing strains of the Bacillus group, in particular of the species Bacillus subtilis, are inter alia, for example, the strains mentioned in WO01/21772

-   -   Bacillus subtilis strain PA 221     -   Bacillus subtilis strain PA 248     -   Bacillus subtilis strain PA 236     -   Bacillus subtilis strain PA 221/pAN429-4     -   Bacillus subtilis strain PA 413-4     -   Bacillus subtilis strain PA 236-1     -   Bacillus subtilis strain PA 340     -   Bacillus subtilis strain PA 377     -   Bacillus subtilis strain PA 365     -   Bacillus subtilis strain PA 377-2     -   Bacillus subtilis strain PA 824-2.

It has been found that microorganisms of the Bacillus group produce D-pantothenic acid in an improved manner after enhancement, in particular over-expression, of one or more of the genes or ORFs, or of the nucleotide sequences which code for these, chosen from the group consisting ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI.

The nucleotide sequences of the genes or open reading frames (ORF) of Bacillus subtilis belong to the prior art and can also be found in the genome sequence of Bacillus subtilis published by Kunst et al. (Nature 390, 249-256 (1997).

-   ybbT-ORF: -   Description: Open reading frame of unknown function, similarity with     phosphoglucomutase -   Reference: Kunst et al.; Nature 390:237-8 (1997) -   Accession No.: Z99104 -   ywkA-ORF -   Description: Open reading frame of unknown function, similarity with     malate dehydrogenase -   Reference: Kunst et al., Nature 390: 249-256 (1997) -   Accession No.: Z99122, Z99123 -   yjmC-ORF -   Description: open reading frame of unknown function, similarity with     malate dehydrogenase from Methanococcus jannaschii -   Reference: Kunst et al., Nature 390: 249-256 (1997) Accession No.:     AF015825, Z99110 -   ytsJ-ORF -   Description: Open reading frame of unknown function, similarity with     malate dehydrogenase (NADP+) from Anas platyrhynchos -   Reference: Abe et al., Microbiology 141, 1433-1442 (1995) -   Accession No.: AP008220 -   mdh gene: -   Description: Malate dehydrogenase -   EC No.: 1.1.1.37 -   Alternative gene name: citH -   Reference: Jin et al.; Journal of Bacteriology 178:560-563 (1996) -   Accession No.: AF008220 -   cysK gene: -   Description: Cysteine synthase A Synonyms: Cysteine synthetase A,     O-acetylserine sulfhydrylase A, O-acetylserine (thiol)-lyase,     (CSase), superoxide inducible protein 11, (SOI11) -   EC No.: 4.2.99.8 -   Reference: Ogasawara et al.; DNA Research 1:1-14 (1994) -   Accession No.: Z99104 -   iolJ gene: -   Description: Fructose 1,6-bisphosphate aldolase (class II) -   Alternative gene names: fbaB, yxdI, alf2, -   EC No.: 4.1.2.13 -   Reference: Yoshida et al.; Microbiology 140:2289-2298 (1994) -   Accession No.: Z99124 -   pdhD gene: -   Description: Dihydrolipoamide dehydrogenase E3 subunit both of the     pyruvate dehydrogenase and of the 2-oxoglutarate dehydrogenase     complex -   Alternative gene names: citL, dld1, aced -   EC No.: 1.8.1.4 -   Reference: Hemila et al., Journal of Bacteriology 172: 5052-5063     (1990) -   Accession No.: AF012285 -   yuiE-ORF: -   Description: Open reading frame of unknown function, similarity with     leucyl aminopeptidase, leucine aminopeptidase (LAP) -   EC No.: 3.4.11.1 -   Reference: Kunst et. al., Nature 390:237-8 (1997) -   Accession No.: Z99120 -   dhaS gene -   Description: Aldehyde dehydrogenase -   Reference: Kunst et al., Nature 390:237-8 (1997) -   Accession No.: AF027868, Z99114 -   adk gene: -   Description: Adenylate kinase, ATP-AMP transphosphorylase,     superoxide inducible protein 16 (SOI16) -   EC No.: 2.7.4.3 -   Reference: Nakamura et al.; Journal of Biochemistry 107:603-607     (1990) -   Accession No.: Z99104 -   yusH-ORF -   Description: Open reading frame of unknown function, similarity with     protein H of the glycine-cleavage system Alternative gene name: gcvH -   Reference: Kunst et al., Nature 390:237-8 (1997) -   Accession No.: Z99120 -   yqhJ-ORF -   Description: Open reading frame of unknown function, similarity with     subunit 1 of glycine dehydrogenase/decarboxylase or of protein P of     the glycine cleavage system -   Alternative gene names: gcvP, gcvPA, gcs1 -   EC No.: 1.4.4.2 -   Reference: Kunst et al., Nature 390: 249-256 (1997) -   Accession No.: Z99116 -   yqhK-ORF -   Description: Open reading frame of unknown function, similarity with     subunit 2 of glycine dehydrogenase/decarboxylase or of protein P of     the glycine cleavage system -   Alternative gene names: gcvP, gcvPB, gcs2 -   EC No.: 1.4.4.2 -   Reference: Kunst et al., Nature 390: 249-256 (1997) -   Accession No.: Z99116 -   yqhI-ORF -   Description: Open reading frame of unknown function, similarity with     the aminomethyl transferase of protein T of the glycine cleavage     system -   Alternative gene names: gcvT, gcsT -   EC No.: 2.1.2.10 -   Reference: Kunst et. al., Nature 390: 249-256 (1997) -   Accession No.: Z99116

The nucleic acid sequences can be found in the databanks of the National Center for Biotechnology Information (NCBI) of the National Library of Medicine (Bethesda, Md., USA), the nucleotide sequence databank of the European Molecular Biologies Laboratories (EMBL, Heidelberg, Germany or Cambridge, UK) or the DNA databank of Japan (DDBJ, Mishima, Japan). The “SubtiList” sequence databank of the Pasteur Institute (Paris, France) can furthermore be used.

The genes or open reading frames described in the text references mentioned can be used according to the invention. Alleles of the genes or open reading frames which result from the degeneracy of the genetic code or due to sense mutations of neutral function can furthermore be used.

In the same way, nucleic acids or polynucleotides which code for proteins or polypeptides which are identical, homologous or similar to the extent of at least 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%, originate from strains of the Bacillus group and have the corresponding function can be used.

To achieve an over-expression, the number of copies of the corresponding genes can be increased, or the promoter and regulation region or the ribosome binding site upstream of the structural gene can be mutated. Expression cassettes which are incorporated upstream of the structural gene act in the same way. By inducible promoters, it is additionally possible to increase the expression in the course of fermentative D-pantothenic acid production. The expression is likewise improved by measures to prolong the life of the m-RNA. Furthermore, the enzyme activity is also increased by preventing the degradation of the enzyme protein. The genes or gene constructs can either be present in plasmids with a varying number of copies, or can be integrated and amplified in the chromosome. Alternatively, an over-expression of the genes in question can furthermore be achieved by changing the composition of the media and the culture procedure.

A strain transformed with one or more plasmid vectors, in particular expression vectors, wherein the plasmid vector(s) carries (carry) at least one of the nucleotide sequences which code for the genes or open reading frames ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI, can be employed in a process according to the invention.

In a further process according to the invention, the ribosome binding sites of one or more of the genes or open reading frames chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI can be optimized.

In a further process according to the invention, an additional promoter can be placed before one or more of the genes or open reading frames chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI.

In a further process according to the invention, at least one further copy of one or more of the genes or open reading frames chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI can be added to the chromosome of the host.

It may furthermore be advantageous for the production of D-pantothenic acid with strains of the Bacillus group, in addition to enhancement of one or more of the genes or open reading frames chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK And yqhI, for one or more of the genes chosen from the group consisting of

-   -   the panE gene which codes for ketopantoate reductase         (WO/0121772)     -   the polypeptide coded by the open reading frame ylbQ or the apbA         gene (Kunst et. al. Nature 20; 390(6657):249-256 (1997);         Accession No. Z99111)     -   the panB gene which codes for ketopantoate hydroxymethyl         transferase (Sorokin et al., Microbiology 142:2005-2016 (1996);         WO01/21772; Accession No.: L47709)     -   the panD gene which codes for aspartate 1-decarboxylase (Sorokin         et al., Microbiology 142:2005-2016 (1996); WO01/21772; Accession         No.: L47709)     -   the panC gene which codes for pantothenate synthetase (Sorokin         et al., Microbiology 142:2005-2016 (1996); WO01/21772; Accession         No.: L47709)     -   the ilvB and ilvN genes which code for acetohydroxy-acid         synthetase (Wipat et. al., Microbiology 142:3067-3078 (1996);         Accession No.: Z75208)     -   the alsS gene which codes for α-acetolactate synthase (Renna et         al., Journal of Bacteriology 175:3863-3875 (1993); Accession         No.: Z93767)     -   the ilvC gene which codes for acetohydroxy-acid isomeroreductase         (Wipat et. al., Microbiology 142:3067-3078 (1996); Accession         No.: Z75208)     -   the ilvD gene which codes for dihydroxy-acid dehydratase         (Sorokin et al., Microbiology 142:2005-2016 (1996); Accession         No.: Z99115)     -   the serA gene which codes for phosphoglycerate dehydrogenase         (Sorokin et al., Molecular Microbiology 10:385-395 (1993);         Accession No.: L47648)     -   the serC gene which codes for phosphoserine amino transferase         (Noback et. al., Microbiology 144:859-875 (1998); Sorokin et         al., Molecular Microbiology 10:385-395 (1993); Accession No.:         Z99109)     -   the open reading frame ywpJ (Presecan et al. Microbiology 143:         3313-3328 (1997); Accession No.: Z83337)     -   the glyA gene which codes for serine hydroxymethyl transferase         (Kunst et. al., Nature 390: 249-256 (1997); Accession No.:         Z99122)     -    to be enhanced, in particular over-expressed, individually or         together.

Finally, it may be advantageous for the production of D-pantothenic acid with strains of the Bacillus group, in addition to enhancement of one or more of the genes or open reading frames chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI, or nucleotide:sequences which code for these, for one or more of the genes or open reading frames chosen from the group consisting of

-   -   the protein coded by ywaA-ORF (Glaser et al., Molecular         Microbiology 10:371-384 (1993); Tobisch et al. Journal of         Bacteriology 179: 496-506 (1997); Accession No. Z49992)     -   the protein coded by ybgE-ORF (Kunst et al., Nature 390, 249-256         (1997); Accession No. Z99105)     -   the ansB gene which codes for L-aspartase (Sun and Seflow,         Journal of Bacteriology 173:3831-3845 (1991); Accession No.:         D84432)     -   the alsD gene which codes for acetolactate decarboxylase (Renna         et al., Journal of Bacteriology 175:3863-3875 (1993); Accession         No.: Z93767)     -   the coaA gene which codes for pantothenic acid kinase, or         yqjS-ORF (Kunst et al., Nature 390, 249-256 (1997); Accession         No.: Z99116)     -   the coaX gene which codes for coax-pantothenic acid kinase, or         yacB-ORF (Kunst et al., Nature 390, 249-256 (1997); Accession         No.: Z99104; WO01/21772)     -   to be attenuated, in particular eliminated or expressed at a low         level, individually or together.

The term “attenuation” in this connection describes the reduction or elimination of the intracellular activity of one or more enzymes (proteins) in a microorganism which are coded by the corresponding DNA, for example by using a weak promoter or using a gene or allele or ORF which codes for a corresponding enzyme (protein) with a low activity or inactivates the corresponding gene or ORF or enzyme (protein) and optionally combining these measures.

The reduction in gene expression can take place by suitable culturing, by genetic modification (mutation) of the signal structures of gene expression or also by the antisense-RNA technique. Signal structures of gene expression are, for example, repressor genes, activator genes, operators, promoters, attenuators, ribosome binding sites, the start codon and terminators.

Mutations which lead to a change or reduction in the catalytic properties or activities of proteins or enzymes are known from the prior art.

Possible mutations are transitions, transversions, insertions and deletions. Depending on the effect of the amino acid exchange on the activity, “missense mutations” or “nonsense mutations” are referred to. Insertions or deletions of at least one base pair in a gene lead to “frame shift mutations”, which lead to incorrect amino acids being incorporated or translation being interrupted prematurely. If a stop codon is formed in the coding region as a consequence of the mutation, this also leads to a premature termination of the translation. Deletions of several codons typically lead to a complete loss of the activity.

By attenuation measures, the activity or concentration of the corresponding protein is in general reduced to 0 to 75%, 0 to 50%, 0 to 25%, 0 to 10% or 0 to 5% of the activity or concentration of the wild-type protein or of the activity or concentration of the protein in the starting microorganism.

It may furthermore be advantageous for the production of D-pantothenic acid, in addition to enhancement of one or more of the genes or open reading frames chosen from the group consisting of ybbT, ywkA, yjmC, ytsJ, mdh, cysK, iolJ, pdhD, yuiE, dhaS, adk, yusH, yqhJ, yqhK and yqhI, to eliminate undesirable side reactions (Nakayama: “Breeding of Amino Acid Producing Microorganisms”, in: Overproduction of Microbial Products, Krumphanzl, Sikyta, Vanek (eds.), Academic Press, London, UK, 1982). Bacteria in which the metabolic pathways which reduce the formation of D-pantothenic acid are at least partly eliminated can be employed in the process according to the invention.

The microorganisms produced according to the invention can be cultured in the batch process (batch culture), the fed batch (feed process) or the repeated fed batch process (repetitive feed process). A summary of known culture methods is described in the textbook by Chmiel (Bioprozesstechnik 1. Einführung die Bioverfahrenstechnik (Gustav Fischer Verlag, Stuttgart, 1991)) or in the textbook by Storhas (Bioreaktoren und periphere Einrichtungen (Vieweg Verlag, Braunschweig/Wiesbaden, 1994)).

The culture medium to be used must meet the requirements of the particular strains in a suitable manner. Descriptions of culture media for various microorganisms are contained in the handbook “Manual of Methods for General Bacteriology” of the American Society for Bacteriology (Washington D.C., USA, 1981). The media described in WO01/21772 can also be used. Sugars and carbohydrates, such as e.g. glucose, sucrose, lactose, fructose, maltose, molasses, starch and cellulose, oils and fats, such as e.g. soya oil, sunflower oil, groundnut oil and coconut fat, fatty acids, such as e.g. palmitic acid, stearic acid and linoleic acid, alcohols, such as e.g. glycerol and ethanol, and organic acids, such as e.g. acetic acid, can be used as the source of carbon. These substances can be used individually or as a mixture.

Organic nitrogen-containing compounds, such as peptones, yeast extract, meat extract, malt extract, corn steep liquor, soya bean flour and urea, or inorganic compounds, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate, can be used as the source of nitrogen. The sources of nitrogen can be used individually or as a mixture.

Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as the source of phosphorus. The culture medium must furthermore comprise salts of metals, such as e.g. magnesium sulfate or iron sulfate, which are necessary for growth. Finally, essential growth substances, such as amino acids and vitamins, can be employed in addition to the above-mentioned substances. Precursors of pantothenic acid, such as aspartate, β-alanine, ketoisovalerate, ketopantoic acid or pantoic acid and optionally salts thereof, can moreover be added to the culture medium. The starting substances mentioned can be added to the culture in the form of a single batch, or can be fed in during the culture in a suitable manner.

Basic compounds, such as sodium hydroxide, potassium hydroxide, ammonia or aqueous ammonia, or acid compounds, such as phosphoric acid or sulfuric acid, can be employed in a suitable manner to control the pH of the culture.

For the preparation of alkaline earth metal salts of pantothenic acid, in particular the calcium salt or magnesium salt, it is equally possible to add the suspension or solution of an inorganic compound containing an alkaline earth metal, such as, for example, calcium hydroxide or MgO, or of an organic compound, such as the alkaline earth metal salt of an organic acid, for example calcium acetate, continuously or discontinuously during the fermentation. For this purpose, the cation necessary for preparation of the desired alkaline earth metal salt of D-pantothenic acid is introduced into the fermentation broth directly in the desired amount, preferably in an amount of 0.95 to 1.1 equivalents.

However, the salts can also be formed after conclusion of the fermentation by addition of the inorganic or organic compounds to the fermentation broth, from which the biomass has optionally been removed beforehand.

Antifoams, such as e.g. fatty acid polyglycol esters, can be employed to control the development of foam. Suitable substances having a selective action, e.g. antibiotics, can be added to the medium to maintain the stability of plasmids. To maintain aerobic conditions, oxygen or oxygen-containing gas mixtures, such as e.g. air, are introduced into the culture. The temperature of the culture is usually 15° C. to 95° C., in particular 15° C. to 70° C., preferably 20° C. to 55° C., very particularly preferably 30° C. to 50° C. or 30° C. to 45° C. Culturing is continued until a maximum of D-pantothenic acid has formed. This target is usually reached within 10 hours to 160 hours.

The D-pantothenic acid or the corresponding salts of D-pantothenic acid contained in the fermentation broth can then be isolated and purified in accordance with the prior art.

It is also possible for the fermentation broths comprising D-pantothenic acid and/or salts thereof preferably first to be freed from all or some of the biomass by known separation methods, such as, for example, centrifugation, filtration, decanting or a combination thereof. However, it is also possible to leave the biomass in its entirety in the fermentation broth. In general, the suspension or solution is preferably concentrated and then worked up to a powder, for example with the aid of a spray dryer or a freeze-drying unit. This powder in then in general converted by suitable compacting or granulating processes, e.g. also build-up granulation, into a coarser-grained, free-flowing, storable and largely dust-free product with a particle size distribution of preferably 20 to 2000 μm, in particular 100 to 1400 μm. In the granulation or compacting it is advantageous to employ conventional organic or inorganic auxiliary substances or carriers, such as starch, gelatin, cellulose derivatives or similar substances, such as are conventionally used as binders, gelling agents or thickeners in foodstuffs or feedstuffs processing, or further substances, such as, for example, silicas, silicates or stearates.

Alternatively, the fermentation product, with or without further of the conventional fermentation constituents, can be absorbed on to an organic or inorganic carrier substance which is known and conventional in feedstuffs processing, such as, for example, silicas, silicates, grits, brans, meals, starches, sugars or others, and/or stabilized with conventional thickeners or binders. Use examples and processes in this context are described in the literature (Die Mühle+Mischfuttertechnik 132 (1995) 49, page 817).

D-Pantothenic acid and/or the desired salt of D-pantothenic acid or a formulation comprising these compounds is optionally added in a suitable process stage during or after the fermentation in order to achieve or establish the content of pantothenic acid desired in the product or the desired salt.

The desired content of pantothenic acid and/or the desired salt is in general in the range from 20 to 80 wt. % (based on the total dry weight).

The concentration of pantothenic acid can be determined with known chemical (Velisek; Chromatographic Science 60, 515-560 (1992)) or microbiological methods, such as e.g. the Lactobacillus plantarum test (DIFCO MANUAL, 10^(th) Edition, p. 1100-1102; Michigan, USA). 

1. A process for the preparation of D-pantothenic acid and/or at least one alkaline earth metal salt thereof, comprising: a) fermenting in a broth microorganisms belonging to the genus Bacillus which produce D-pantothenic acid, wherein a cysteine synthase (cysK) polynucleotide from Bacillus is over-expressed in the microorganisms, and b) concentrating the D-pantothenic acid and/or at least one salt thereof in the fermentation broth or in the microorganisms.
 2. A process according to claim 1, wherein the fermentation is carried out in the presence of at least one alkaline earth metal salt, said at least one alkaline earth metal salt being added continuously or discontinuously, thereby obtaining a product comprising at least one alkaline earth metal salt of D-pantothenic acid.
 3. A process according to claim 1, wherein the microorganisms are of the species Bacillus subtilis.
 4. A process according to claim 1, wherein at least one polynucleotide from Bacillus selected from the group consisting of: 4.1 a panE polynucleotide which encodes ketopantoate reductase, 4.2 an open reading frame ylbQ, 4.3 a panB polynucleotide which encodes ketopantoate hydroxymethyl transferase, 4.4 a panD polynucleotide which encodes aspartate decarboxylase, 4.5 a panC polynucleotide which encodes pantothenate synthetase, 4.6 a ilvB and ilvN polynucleotides which encode acetohydroxy acid synthetase, 4.7 a alsS polynucleotide which encodes α-acetolactate synthase, 4.8 a ilvC polynucleotide which encodes acetohydroxy-acid isomeroreductase, 4.9 a ilvD polynucleotide which encodes dihydroxy-acid dehydratase, 4.10 a serA polynucleotide which encodes phosphoglycerate dehydrogenase, 4.11 a serC polynucleotide which encodes phosphoserine amino transferase, 4.12 a polynucleotide which encodes the open reading frame ywpJ, and 4.13 a glyA polynucleotide which encodes serine hydroxymethyl transferase, is over-expressed.
 5. A process according to claim 1, wherein the over-expression is achieved by carrying out at least one measure selected from the group consisting of use of a plasmid vector, optimization of the ribosome binding site, use of an additional promoter and incorporation of at least one additional gene copy.
 6. A process according to claim 1, further comprising: after said fermentation, isolating the D-pantothenic acid and/or at least one salt thereof from the fermentation broth.
 7. A process according to claim 2, wherein the at least one alkaline earth metal salt is added in stoichiometric amounts equivalent to the D-pantothenic acid formed.
 8. A process for the preparation of a feedstuffs additive comprising D-pantothenic acid and/or at least one salt thereof, comprising: a) fermenting in a fermentation broth microorganisms belonging to the genus Bacillus which produce D-pantothenic acid, wherein a cysteine synthase (cysK) polynucleotide from Bacillus is over-expressed in the microorganisms, b) optionally concentrating D-pantothenic acid and/or at least one salt thereof in the fermentation broth or in the microorganisms, c) optionally separating off at least a portion of a biomass and/or a portion of other constituents from said fermentation broth containing the D-pantothenic acid and/or at least one salt thereof, d) optionally concentrating said fermentation broth after step c), e) converting the D-pantothenic acid and/or at least one salt thereof in said fermentation broth into a feedstuffs additive, f) optionally converting said feedstuffs additive into a free-flowing form, and g) optionally providing said free flowing feedstuffs additive with a particle size distribution of 20 to 2000 μm.
 9. A process for the preparation of a feedstuffs additive comprising at least one alkaline earth metal salt of D-pantothenic acid, comprising: a) fermenting in a fermentation broth microorganisms belonging to the genus Bacillus which produce D-pantothenic acid, wherein a cysteine synthase (cysK) polynucleotide from Bacillus is over-expressed in the microorganisms, b) optionally concentrating D-pantothenic acid and/or at least one salt thereof in the fermentation broth or in the microorganisms, c) optionally separating off at least a portion of a biomass and/or a portion of other constituents from said fermentation broth containing the D-pantothenic acid and/or at least one salt thereof, d) adding at least one alkaline earth metal compound to said fermentation broth to produce an alkaline earth metal salt of said D-pantothenic acid, e) converting the alkaline earth metal salt of the D-pantothenic acid in said fermentation broth into a feedstuffs additive, f) optionally converting said feedstuffs additive into a free-flowing form, and g) optionally providing said free flowing feedstuffs additive with a particle size distribution of 20 to 2000 μm.
 10. A process according to claim 9, wherein an animal feedstuffs additive with a desired particle size is obtained by a) drying and compacting, or b) spray drying, or c) spray drying and granulation, or d) spray drying and build-up granulation.
 11. A process according to claim 9, wherein the alkaline earth metal in the at least one salt and in the at least one alkaline earth metal compound is calcium or magnesium.
 12. A process according to claim 9, further comprising: converting the animal feedstuffs additive into powder or granule form. 